Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein

Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.     

Immunodominance and functional activities of antibody responses to inactivated West Nile virus and recombinant subunit vaccines in mice

Factors controlling the dominance of antibody responses to specific sites in viruses and/or protein antigens are ill defined but can be of great importance for the induction of potent immune responses to vaccines. West Nile virus and other related important human-pathogenic flaviviruses display the major target of neutralizing antibodies, the E protein, in an icosahedral shell at the virion surface. Potent neutralizing antibodies were shown to react with the upper surface of domain III (DIII) of this protein. Using the West Nile virus system, we conducted a study on the immunodominance and functional quality of E-specific antibody responses after immunization of mice with soluble protein E (sE) and isolated DIII in comparison to those after immunization with inactivated whole virions. With both virion and sE, the neutralizing response was dominated by DIII-specific antibodies, but the functionality of these antibodies was almost four times higher after virion immunization. Antibodies induced by the isolated DIII had an at least 15-fold lower specific neutralizing activity than those induced by the virion, and only 50% of these antibodies were able to bind to virus particles. Our results suggest that immunization with the tightly packed E in virions focuses the DIII antibody response to the externally exposed sites of this domain which are the primary targets for virus neutralization, different from sE and isolated DIII, which also display protein surfaces that are cryptic in the virion. Despite its low potency for priming, DIII was an excellent boosting antigen, suggesting novel vaccination strategies that strengthen and focus the antibody response to critical neutralizing sites in DIII.     

Inactivated SARS-CoV vaccine prepared from whole virus induces a high level of neutralizing antibodies in BALB/c mice

We tested the ability of inactivated SARS-CoV vaccine to induce neutralizing antibodies in BALB/c mice. The inactivated vaccine was prepared by SARS-CoV virus propagation in Vero cells, with subsequent beta-propiolactone inactivation and Sepharose 4FF column chromatography purification. One hundred forty BALB/c female mice were divided into seven groups of 20 mice each. Of the seven groups, three groups were inoculated with 0.1, 1, and 3 microg of the vaccine without adjuvant while three other groups were inoculated at the same three dosages of vaccine with aluminum hydroxide as adjuvant, respectively. The remaining group was set up as a blank control. Each mouse was inoculated twice at an interval of 3 weeks. One week after the second immunization, mice sera were collected to detect serum neutralizing antibodies. An assay for determining neutralizing antibody titers was developed. The results can be summarized as follows: (1) higher dosages of vaccine induced higher levels of neutralizing antibody titer; (2) the level of neutralizing antibodies induced by the inoculation with aluminum hydroxide adjuvant was slightly higher than that without adjuvant, but the difference was not statistically significant.     

Dissection of Antibody Specificities Induced by Yellow Fever Vaccination

The live attenuated yellow fever (YF) vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III) and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity) as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual-specific factors on immunodominance in humoral immune responses.     

Monoclonal antibodies against hen's egg ovomucoid

Two monoclonal antibodies (mAb 23E5 and 32A8) to hen's egg ovomucoid (OM), which causes hen's egg allergy and has trypsin inhibitory activity, were prepared and purified. Their affinity to the three separate domains of the ovomucoid, which are homologous in primary structure and are designated as DI, DII, and DIII, was studied by a competitive radioimmunoassay. MAb 23E5 bound to OM more efficiently than to DI, DII, or DIII-2 (with carbohydrate), but reacted with DIII-1 (free from carbohydrate) more efficiently than with OM. Except for the binding to OM, mAb 32A8 bound to DIII-2 most efficiently and to DIII-1 least efficiently, suggesting that this antibody recognized the carbohydrate moiety of DIII. MAb 32A8 inhibited the trypsin inhibitory activity of OM, whereas mAb 23E5 had no effect on it. These monoclonal antibodies should be useful for analyzing the antigenic determinants and trypsin inhibitory activity of ovomucoid.     

Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile virus infection

The domain III of the West Nile virus (WNV) envelope glycoprotein (E) was shown to serve as virus attachment domain to the cellular receptor, and neutralizing Abs have been mapped to this specific domain. In this study, domain III of the WNV E protein (WNV E DIII) was expressed as a recombinant protein and its potential as a subunit vaccine candidate was evaluated in BALB/C mice. Immunization of WNV E DIII protein with oligodeoxynucleotides (CpG-DNA) adjuvant by i.p. injection was conducted over a period of 3 wk. The immunized mice generated high titer of WNV-neutralizing Abs. Murine Ab against WNV E DIII protein was also capable of neutralizing Japanese encephalitis virus. The IgG isotypes generated were predominantly IgG2a in the murine sera against the recombinant protein. Splenocyte cultures from the mice coadministrated with WNV E DIII protein and CpG secreted large amounts of IFN-gamma and IL-2 and showed proliferation of T cells in the presence of WNV E DIII protein. Overall, this study highlighted that recombinant WNV E DIII protein delivered in combination with CpG adjuvant to mice generated a Th1 immune response type against WNV and can serve as a potential vaccine to prevent WNV infection.     

Redundancy and plasticity of neutralizing antibody responses are cornerstone attributes of the human immune response to the smallpox vaccine

The smallpox vaccine is widely considered the gold standard for human vaccines, yet the key antibody targets in humans remain unclear. We endeavored to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans which could be used as a diagnostic serological marker of protective humoral immunity induced by the smallpox vaccine (vaccinia virus [VACV]). We have instead found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show that H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of immunoglobulin G (IgG) specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV. Comparable results were obtained for human (and mouse) anti-L1 IgG and even for anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen but instead by driving development of neutralizing antibodies to multiple viral proteins, resulting in a "safety net" of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose that this is a fundamental attribute of the smallpox vaccine.     

Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection‐enhancing antibody response

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection‐enhancing activities at sub‐neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM‐E) gene‐based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM‐E gene backbone showed high neutralizing antibody titers with less DENV infection‐enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.     

Induction of epitope-specific neutralizing antibodies against West Nile virus

Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies.     

The Development of Therapeutic Antibodies That Neutralize Homologous and Heterologous Genotypes of Dengue Virus Type 1

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential.     

Potent Dengue Virus Neutralization by a Therapeutic Antibody with Low Monovalent Affinity Requires Bivalent Engagement

We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface.     

An mRNA vaccine encoding Chikungunya virus E2-E1 protein elicits robust neutralizing antibody responses and CTL immune responses

Arthropod-borne chikungunya virus (CHIKV) infection can cause a debilitating arthritic disease in human. However, there are no specific antiviral drugs and effective licensed vaccines against CHIKV available for clinical use. Here, we developed an mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing CHIKV E2-E1 antigen, and compared its immunogenicity with soluble recombinant protein sE2-E1 antigen expressed in S2 cells. For comparison, we first showed that recombinant protein antigens mixed with aluminum adjuvant elicit strong antigen-specific humoral immune response and a moderate cellular immune response in C57BL/6 mice. Moreover, sE2-E1 vaccine stimulated 12-23 folds more neutralizing antibodies than sE1 vaccine and sE2 vaccine. Significantly, when E2-E1 gene was delivered by an mRNA-LNP vaccine, not only the better magnitude of neutralizing antibody responses was induced, but also greater cellular immune responses were generated, especially for CD8⁺ T cell responses. Moreover, E2-E1-LNP induced CD8⁺ T cells can perform cytotoxic effect in vivo. Considering its better immunogenicity and convenience of preparation, we suggest that more attention should be placed to develop CHIKV E2-E1-LNP mRNA vaccine.     

Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.     

Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type 2 envelope protein recognize adjacent epitopes

Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.     

登革病毒四型联合重组包膜蛋白III区的表达和免疫原性鉴定

登革热在全球范围内广泛流行,但是目前为止却仍然没有疫苗上市,疫苗的开发迫在眉睫。抗体依赖增强感染效应是登革病毒疫苗开发中遇到的一个瓶颈问题。研究表明登革病毒的包膜蛋白III区能够介导中和抗体产生,且诱导产生较少的交叉抗体或无交叉抗体,能够大大减弱抗体依赖增强感染效应,因而是登革热重组蛋白疫苗的首选靶标。通过酵母密码子优化后合成同时包含4种血清型登革病毒包膜蛋白III区的四价联合DV EDIII蛋白序列,随后构建酵母表达质粒,并获得酵母表达菌株,经诱导后四联DV EDIII蛋白获得高效表达。通过Western blot、ELISA检测及蛋白质免疫原性鉴定,结果表明登革病毒四联DV EDIII蛋白表达质粒构建成功,重组蛋白在毕赤酵母获得高效表达,免疫小鼠后能够介导产生较高水平的血清效价。这表明已获得了能引起有效免疫反应的四型登革病毒EDIII蛋白,为登革病毒疫苗的研究提供了良好的基础。     

Mutagenesis of the DI/DIII linker in dengue virus envelope protein impairs viral particle assembly

The dengue virus (DV) envelope (E) protein is important in mediating viral entry and assembly of progeny virus during cellular infection. Domains I and III (DI and DIII, respectively) of the DV E protein are connected by a highly conserved but poorly ordered region, the DI/DIII linker. Although the flexibility of the DI/DIII linker is thought to be important for accommodating the structural rearrangements undergone by the E protein during viral entry, the function of the linker in the DV infectious cycle is not well understood. In this study, we performed site-directed mutagenesis on conserved residues in the DI/DIII linker of the DV2 E protein and showed that the resulting mutations had little or no effect on the entry process but greatly affected virus assembly. Biochemical fractionation and immunofluorescence microscopy experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the DI/DIII linker mutants express the DV structural proteins at the sites of particle assembly near the ER but fail to form infectious particles. This defect is not due to disruption of E's interaction with prM and pr in immature and mature virions, respectively. Serial passaging of the DV2 mutant E-Y299F led to the identification of a mutation in the membrane-proximal stem region of E that fully compensates for the assembly defect of this DI/DIII linker mutant. Together, our results suggest a critical and previously unidentified role for the E protein DI/DIII linker region during the DV2 assembly process.     

Zika virus structural biology and progress in vaccine development

The growing number of zika virus (ZIKV) infections plus a 20-fold increase in neonatal microcephaly in newborns in Brazil have raised alarms in many countries regarding the threat to pregnant women. Instances of microcephaly and central nervous system malformations continue to increase in ZIKV outbreak regions. ZIKV is a small enveloped positive-strand RNA virus belonging to the Flavivirus genus of the Flaviviridae family. High-resolution ZIKV structures recently identified by cryo-electron microscopy indicate that the overall ZIKV structure is similar to those of other flaviviruses. With its compact surface, ZIKV is more thermally stable than the dengue virus (DENV). ZIKV E proteins have a characteristic “herringbone” structure with a single glycosylation site. The ZIKV E protein, the major protein involved in receptor binding and fusion, is formed as a head-to-tail dimer on the surfaces of viral particles. The E monomer consists of three distinct domains: DI, DII, and DIII. The finger-like DII contains a fusion loop (FL) that is inserted into the host cell endosomal membrane during pH-dependent conformational changes that drive fusion. Quaternary E:E dimer epitopes located at the interaction site of prM and E dimers can be further divided into two dimer epitopes. To date, more than 50 ZIKV vaccine candidates are now in various stages of research and development. Candidate ZIKV vaccines that are currently in phase I/II clinical trials include inactivated whole viruses, recombinant measles viral vector-based vaccines, DNA and mRNA vaccines, and a mosquito salivary peptide vaccine. Stabilized forms of ZIKV E:E dimer proteins have been successfully obtained either by introducing additional inter-subunit disulfide bond(s) in DII or via the direct assembly of E:E dimer proteins by immobilization with monomeric E proteins. The VLP-based approach is another alternative method for presenting native E:E dimer antigens among the vaccine components. Several forms of ZIKV VLPs have been reported featuring the co-expression of the prM-E, prM-E-NS1, C-prM-E, and NS2B/NS3 viral genes in human cells. To minimize the effect of the cross-reactive ADE-facilitating antibodies between ZIKV and DENV, several novel mutations have been reported either in or near the FL of DII or DIII to dampen the production of cross-reactive antibodies. Future ZIKV vaccine design efforts should be focused on eliciting improved neutralizing antibodies with a reduced level of cross-reactivity to confer sterilizing immunity.     

Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes

Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.     

Adjuvant effects of dimethyl dioctadecyl ammonium bromide, complete Freund's adjuvant and aluminium hydroxide on neutralizing antibody, antibody-isotype and delayed-type hypersensitivity responses to Semliki Forest virus in mice.

Outbred mice were inoculated subcutaneously with inactivated Semliki Forest virus (SFV) in saline and combinations of the virus with complete Freund's adjuvant (CFA) aluminium hydroxide (Al) and dimethyl dioctadecyl ammonium bromide (DDA). The immune response was evaluated for delayed-type hypersensitivity, for total ELISA antibodies and antibody-isotypes and for neutralizing antibodies. DDA was the most efficient adjuvant in inducing DTH, CFA the second and Al induced a DTH response that was only slightly higher (statistically not significant) than that induced by the inactivated virus without adjuvants. All adjuvants enhanced the production of ELISA antibodies to similar levels. However, the levels of neutralizing antibodies induced were low in mice which were inoculated with the inactivated SFV alone or mixtures of the virus with Al. DDA induced high levels of neutralizing antibodies and CFA induced intermediate levels. The pattern of antibody-isotypes induced by DDA and CFA was different from the pattern induced by the inactivated virus or by the virus mixed with Al: DDA and CFA induced low amounts of IgG1 antibodies and relatively higher amounts of IgG2a and IgG2b antibodies while the inactivated virus and the mixture of the virus with Al induced higher proportions of IgG1 antibodies. In sera from convalescent mice the majority of antibody activity resided in the IgG2a and IgG2b immunoglobulin subclasses, while IgG1 antibodies were undetectable.     

Glycosylation of immunodominant linear epitopes in the carboxy-terminal region of the caprine arthritis-encephalitis virus surface envelope enhances vaccine-induced type-specific and cross-reactive neutralizing antibody responses

This study evaluated type-specific and cross-reactive neutralizing antibodies induced by immunization with modified surface glycoproteins (SU) of the 63 isolate of caprine arthritis-encephalitis lentivirus (CAEV-63). Epitope mapping of sera from CAEV-infected goats localized immunodominant linear epitopes in the carboxy terminus of SU. Two modified SU (SU-M and SU-T) and wild-type CAEV-63 SU (SU-W) were produced in vaccinia virus and utilized to evaluate the effects of glycosylation or the deletion of immunodominant linear epitopes on neutralizing antibody responses induced by immunization. SU-M contained two N-linked glycosylation sites inserted into the target epitopes by R539S and E542N mutations. SU-T was truncated at 518A, upstream from the target epitopes, by introduction of termination codons at 519Y and 521Y. Six yearling Saanen goats were immunized subcutaneously with 30 microg of SU-W, SU-M, or SU-T in Quil A adjuvant and boosted at 3, 7, and 16 weeks. SU antibody titers determined by indirect enzyme-linked immunosorbent assay demonstrated anamnestic responses after each boost. Wild-type and modified SU-induced type-specific CAEV-63 neutralizing antibodies and cross-reactive neutralizing antibodies against CAEV-Co, a virus isolate closely related to CAEV-63, and CAEV-1g5, an isolate geographically distinct from CAEV-63, were determined. Immunization with SU-T resulted in altered recognition of SU linear epitopes and a 2.8- to 4.6-fold decrease in neutralizing antibody titers against CAEV-63, CAEV-Co, and CAEV-1g5 compared to titers of SU-W-immunized goats. In contrast, immunization with SU-M resulted in reduced recognition of glycosylated epitopes and a 2.4- to 2.7-fold increase in neutralizing antibody titers compared to titers of SU-W-immunized goats. Thus, the glycosylation of linear immunodominant nonneutralization epitopes, but not epitope deletion, is an effective strategy to enhance neutralizing antibody responses by immunization.